Lecture 5 16.11.2017 – FALL 2017 - PowerPoint Presentation

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Lecture 516.11.2017 – FALL 2017

Protein Isolation and Quantification

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How to isolate total protein

Lyse

the cell

Solubilize

the protein

For

solubilize

membrane protein, we have to use detergent in protein extraction buffer.

A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. 

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The general detergent used in the protein extraction buffer

Nonionic Detergents (milder)

Triton X100: Break lipid-lipid interaction and lipid-protein interaction

Anionic Detergent

SDS: Protein-Protein interaction

Sodium

Deoxycholate

: Protein-Protein interaction

LYSIS BUFFER

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Important Factors to be Considered When Choosing Lysis Buffer

pH

ionic strength

usage of detergent

preventative measure for proteolytic processes

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Proteases inhibitor Upon lysis of the cell, proteases are released into the lysate

What are proteases?

Where are the proteases from when isolating the protein?

What are the proteases?

Enzymes that break peptide bonds between amino acids of proteins.

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Where are the proteases from when isolating the protein?

Animal cells:

Lysosomes

, contain a large variety of hydrolytic

enzmyes

that degrade proteins other substances.

Plant cells: Vacuole, many hydrolytic enzymes found in vacuole resemble those present in lysosomes of animal cells other

organnells

also have proteases.

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How to prevent the proteins from degradation by protease?

The protein isolation is carried out at low temperature to minimize the activities of the proteases.

The further optimize the results, we use the

proteases inhibitors

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Often used chemical protease inhibitors in protein isolation

EDTA: Chelating the Ca

+2

PMSF: a general serine protease inhibitor. It is the most common inhibitor used in protein purification. Soluble in

isopropanol

.

The protease inhibitors cocktail: a mixture of several protease inhibitors with broad specify.

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The protein quantification

UV 280 absorption

Colorimetric methods

Biuret

Lowry

Bradford

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UV absorption method

The amino acid tryptophan, tyrosine and

phenlyalenine

absorb light in the UV wavelength.

Since the absorption is proportional to concentration, this is a useful way to quantitates protein concentration (for protein containing

Trp

)

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Disadvantages of UV absorption method

If some protein do not contain the amino acids, it will not absorb UV light.

Nucleic acid (DNA, RNA)contaminant will also absorb UV light.

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Calorimetric methods

We can modify the protein sample with appropriate reagents so as to produce a color reaction and measure protein concentration using a spectrophotometer.

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Advantages of colorimetric methods

cheaper

cuvette

(cheap glass or plastic versus quartz

quartz

)

Not

contamining

absorbance from nucleic acid.

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Colorimetric methods I: Bradford Method

A dye known as

coomassie

Brilliant Blue was developed for the textile industry. It was noticed to stain skin as well as the textile.

This dye (which normally absorbs at 465 nm) binds to the proteins and to absorb strongly at 595 nm)

The assay is sensitive but somewhat non-linear.

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Lowery method

A widely used method of measuring protein concentration.

A colorimetric assay.

Amount of the blue color proportional to amount of protein.

Absorbance read using 500-750nm light.

Lowery at all. 1951

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Lowry Method

Two reaction make the blue color develop:

Reactions of

copper ion with the peptide bonds

under alkaline conditions with the oxidation of aromatic protein residues.

Folin-ciocatleu

reagent is used.

Concentration of the reduced

Folin

reagent is measured by absorbance at 750nm.

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Making a strand curve with BSA (bovine serum albumin)

A graph that correlates Absorbance with protein concentration

Standard Curve generated by doing a Lowry Assay on protein solution of known concentration

Standard curve must be done each of the time unknowns are being tested

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Standard Curve

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Using Standard Curve

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The SDS-PAGE

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Gels are cast by polymerizing a solution of

acrylamide

monomers into

polyacrylamide

chains.

Gel pore size can be varied by adjusting the concentrations of

polyacrylamide

.

Smaller proteins migrate faster than larger proteins through

th

e gel

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SDS (Sodium dodecyl sulfate)

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SDS

SDS disturbs some of the

noncovalent

interactions

that stabilize protein quaternary and tertiary structures, facilitates

denaturation

.

SDS also has a negative electrical charge and binds to proteins in a constant mass ratio 1:4:1, so the total amount of detergent bound is directly proportional to the molecular weight of the protein.

The coating of negatively charged SDS overwhelms the inherent charges of protein molecules and gives them uniform charge to mass ratio

This allows proteins to be separated on the basis of their relative sizes.

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How about covalent link?

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Heating your samples at 99C completed denaturation of the protein molecules, ensuring that they were in completely linear form. This allowed SDS to bind all regions of each protein equally.

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Protein Loading Buffer

Protein gel loading buffer contains

Tris

buffer to maintain constant

pH.

Glycerol

to increase sample density.

The strong ionic detergent SDS (sodium

dodecylsulfate

)

Β-mercaptoethanol a reducing agent. Beta mercaptoethanol eliminates disulfide bonds in protein by reducing them (adding hydrogen atoms).

Heating

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Stacking Gel

To obtain optimum resolution of proteins, a “stacking” gel is poured over the top of the resolving gel.

The stacking gel

Lower concentration of

acrylamide

(larger pore size)

Lower pH

Different ionic content

This allows the proteins in a lane to be concentrated into a tight band before entering the running or resolving gel

Produces a gel with tighter or better separated protein bands.

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Gel Staining

Once protein fractionated by electrophoresis, to make them visible, staining with the material that will bind to proteins but not

polyacrylamide

.

The most common one: staining with

Coomassie

Blue.

This is a dye that binds most proteins uniformly based on interactions with the carbon-nitrogen backbone.

The dye is dissolved in a solution that contains both methanol and acetic acid.

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Gel Drying Frame

OCTAFRAME

 is a set of rigid plastic frames to support SDS-PAGE gels between two sheets of cellophane for drying on the bench. The drying is a few hours in gentle stream of air

It is important to remove all the air bubbles from between the two sheets of gel drying films.

Air bubbles may cause the gel crack during drying.

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Modern Technique: HPLC

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Uses of HPLC

This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.

In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

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Picture of an HPLC column

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WHAT AFFECTS SYSTEM

Column Parameters

Column Material

Deactivation

Stationary Phase

Coating Material

Instrument Parameters

Temperature

Flow

SignalSample Sensitivity

Detector

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Several column types(can be classified as )

Normal phase

Reverse phase

Size exclusion

Ion exchange

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Normal phase

In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute

.

For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

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Reverse phase

In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample.

Typical

stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

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Size exclusion

In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size.

Small

molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

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Ion exchange

In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase

.

Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites

.